Photonic crystals, also commonly referred to as photonic bandgap structures, are periodic dielectric or metallic structures exhibiting a spatially periodic variation in refractive index that forbids propagation of certain frequencies of incident electromagnetic radiation. The photonic band gap of a photonic crystal refers to the range of frequencies of electromagnetic radiation for which propagation through the structure is prevented. The photonic band gap phenomenon may be conceptualized as complete reflection of incident electromagnetic radiation having selected frequencies due to interaction with the periodic structural domains of a photonic crystal. The spatial arrangement and refractive indices of these structural domains generate photonic bands gaps that inhibit propagation of electromagnetic radiation centered about a particular frequency. Background information on photonic crystals include the following references: (1) Joanopoulus et al., “Photonic Crystals Molding the Flow of Light”, Princeton University Press, 1995; (2) A. Birner, et al., “Silicon-Based Photonic Crystals”, Advanced Materials, Volume 13, Issue 6, Pages 377-388; and (3) Steven G. Johnson, and John D. Joannopoulos, “Photonic Crystals: The Road from Theory to Practice”, Springer, 2002.
Photonic crystals provide an electromagnetic analog to electron-wave behavior observed in crystals wherein electron-wave concepts, such as dispersion relations, Bloch wave functions, van Hove singularities and tunneling, having electromagnetic counterparts in photonic crystals. In semiconductor crystals, for example, an electronic band gap of energy states for which electrons are forbidden results from a periodic atomic crystalline structure. By analogy, in a photonic crystal, a photonic band gap of forbidden energies (or wavelengths/frequencies) of electromagnetic radiation results from a periodic structure of a dielectric material where the periodicity is of a distance suitable to interact with incident electromagnetic radiation.
Selection of the physical dimensions, refractive indices and spatial distribution of periodic structural components (“surface grating” herein) of a photonic crystal provides an effective means of designing a photonic crystal a photonic band gap with a selected frequency distribution. If the periodicity and symmetry of the crystal and the dielectric constants of the materials used are chosen appropriately, the photonic crystal will selectively couple energy at particular wavelengths, while excluding others. One-dimensional, two-dimensional and three-dimensional photonic crystals have been fabricated providing complete or at least partial photonic band having selected frequency distributions gaps in one or more directions. Photonic crystals have also been fabricated having selected local disruptions (e.g., missing or differently-shaped portions of the structural domains of periodic array) in their periodic structure, thereby generating defect or cavity modes with frequencies within a forbidden bandgap of the crystal. Photonic crystals having specific defects are of particular interest because they provide optical properties useful for controlling and manipulating electromagnetic radiation, such as the ability to provide optical confinement and/or wave guiding with very little, or substantially no, radiative losses. U.S. Pat. No. 6,990,259 to Cunningham describes a “defect” biosensor in greater detail. The content of the '259 patent is incorporated by reference herein.
As diffraction and optical interference processes give rise to the photonic band gap phenomenon, the periodicity of photonic crystal structures is typically on the order of the wavelength of incident electromagnetic radiation. Accordingly, photonic crystals for controlling and manipulating visible and ultraviolet electromagnetic radiation typically comprise dielectric or metallic structures with periodic structural domains having submicron physical dimensions on the order of 100's of nanometers. A number of fabrication pathways for making periodic structures having these physical dimensions have been developed over the last decade, including micromachining and nanomachining techniques (e.g., lithographic patterning and dry/wet etching, electrochemical processing etc.), colloidal self assembly, replica molding, layer by-layer assembly and interference lithography. Advances in these fabrication techniques have enabled fabrication of one-dimensional, two-dimensional and three-dimensional photonic crystals from a range of materials including dielectric crystals, metals, polymers and colloidal materials.
The applications of photonic crystal sensors are numerous, including integration with lasers to inhibit or enhance spontaneous emission, waveguide angle steering devices, and as narrowband optical filters. A photonic crystal structure geometry can be designed to concentrate light into extremely small volumes and to obtain very high local electromagnetic field intensities.
In order to adapt a photonic crystal device to perform as a biosensor, some portion of the structure must be in contact with a test sample. By attaching biomolecules or cells to the portion of the photonic crystal where the locally confined electromagnetic field intensity is greatest, the resonant coupling of light into the crystal is modified, so the reflected/transmitted output is tuned. The highly confined electromagnetic field within a photonic crystal structure provides high sensitivity and a high degree of spatial resolution consistent with their use in imaging applications, much like fluorescent imaging scanners.
For example, photonic crystals with subwavelength periodic grating structures have been developed to reflect only a very narrow band of wavelengths when illuminated with white light. To create a biosensor, a photonic crystal may be optimized to provide an extremely narrow resonant mode whose wavelength is particularly sensitive to modulations (i.e., shifts) induced by the deposition of biochemical material on its surface. In typical practice, a photonic crystal sensor consists of a low refractive index plastic material with a periodic surface structure that is coated with a thin layer of high refractive index dielectric material. The sensor is measured by illuminating the surface with white light, and collecting the reflected light with a non-contact optical fiber probe, where several parallel probes can be used to independently measure shifts in the peak wavelength of reflected light (“PWV”) at different locations on the sensor. The biosensor design enables a simple manufacturing process to produce sensor sheets in continuous rolls of plastic film that are hundreds of meters in length. The mass manufacturing of a biosensor structure that is measurable in a non-contact mode over large areas enables the sensor to be incorporated into single-use disposable consumable items such as 96, 384, and 1536-well standard microplates, thereby making the sensor compatible with standard fluid handling infrastructure employed in most laboratories. In these cases, the photonic crystal is manufactured in separate manufacturing operation, and then, in a second step, glued or otherwise adhered to a bottomless microplate. The wells of the microplates provide a reservoir by which a fluid sample can be introduced onto the photonic crystal surface.
The sensor operates by measuring changes (shifts) in the wavelength of reflected light (“PWV”) as biochemical binding events take place on the surface. For example, when a protein is immobilized on the sensor surface, an, increase in the reflected wavelength is measured when a complementary binding protein is exposed to the sensor. Using low-cost components, the readout instrument is able to resolve protein mass changes on the surface with resolution less than 1 pg/mm2. While this level of resolution is sufficient for measuring small-molecule interactions with immobilized proteins, the dynamic range of the sensor is large enough to also measure larger biochemical entities including live cells, cell membranes, viruses, and bacteria. A sensor measurement requires about 20 milliseconds, so large numbers of interactions can be measured in parallel, and kinetic information can be gathered. The reflected wavelength of the sensor can be measured either in “single point mode” (such as for measuring a single interaction within a microplate), or an imaging system can be used to generate an image of a sensor surface with <9 μm resolution. The “imaging mode” has been used for applications that increase the overall resolution and throughput of the system such as label-free microarrays, imaging plate reading, self-referencing microplates, and multiplexed spots/well.
Given substantial advances in their fabrication and their unique optical properties, photonic crystal-based sensors have been recently developed for a range of biosensing applications. To operate as a biosensor, a photonic crystal is provided in a configuration such that its active area is exposed to a fluid containing analytes for detection. The presence of analyte proximate to the photonic crystal sensor modulates the resonant coupling of light into the crystal, thereby resulting in a measurable change in the wavelength distribution of electromagnetic radiation transmitted, scattered or reflected by the crystal resulting from changes in the photonic band gap of the crystal. The highly localized nature of the confined electromagnetic field generated by the crystal ensures that that detection via photonic crystal based sensors is restricted to a probe region proximate to (e.g., 100-400 nanometers) the active area of the sensor. In typical sensing applications, a read out system is used wherein polarized electromagnetic radiation having a selected wavelength distribution is provided to the photonic crystal and subsequently reflected or transmitted electromagnetic radiation is frequency analyzed by an appropriate photodetector, such as a spectrometer in combination with an appropriate detector. By observing and/or quantifying the change in wavelength distribution resulting from interaction of the fluid and the photonic crystal, analytes in the probe region are detected and/or analyzed.
Biosensors incorporating photonic crystal structures are described in the following references which are hereby incorporate by reference: U.S. Pat. Nos. 7,118,710, 7,094,595, 7,023,544, and 6,990,259; and Cunningham, B. T., P. Li, B. Lin and J. Pepper, Colorimetric Resonant Reflection as a Direct Biochemical Assay Technique, Sensor and Actuators B, 2002, 81, pgs 316-328; and Cunningham, B. T. J. Qiu, P. Li, J. Pepper and B. Hugh, A Plastic Calorimetric Resonant Optical Biosensor for Multiparallel Detection of Label 10 Free Biochemical Interactions, Sensors and Actuators B, 2002, 85, pgs 219-226.
Advantages provided by photonic crystals for biosensing include the ability to detect and characterize a wide range of materials, including peptides, proteins, oligonucleotides, cells, bacteria and virus particles, without the use of labels, such as fluorescent labels and radioligands, or secondary reporter systems. Direct detection provided by photonic crystal sensing enhances easy of implementation of these techniques by eliminating labor intensive processing required to synthetically link and/or read out a label or reporter system. This beneficial aspect of photonic crystal-based sensing also eliminates a significant source of experimental uncertainty arising from the influence of a label or reporter system on molecular conformation, reactivity, bioactivity and/or kinetics; and eliminates problems arising from liquid phase fluorescence quenching processes. Photonic crystal based sensors are also compatible with functionalization, for example by incorporation of biomolecules and/or candidate therapeutic molecules bound to the surface of the active area of the photonic crystal structure; a capability which is particularly attractive for selectively detecting specific target molecules for screening and biosensing applications. Other benefits provided by photonic crystal approaches to biosensing include: (i) good sensitivity and image resolution; (ii) compatibility with relatively straightforward optical readout systems, (iii) and the ability to provide highly localized detection useful for multichannel systems having a high area density sensors are emerging as a major tool for selective biochemical detection and analysis in diverse fields including genomics, proteomics, pharmaceutical screening and biomedical diagnostics.
In current practice, photonic crystal biosensors and the associated larger-scale fluid containment features (such as wells or channels) are typically fabricated separately and subsequently integrated via alignment and bonding processes. Given the submicron scale of features of the photonic crystal and micron or larger scale physical dimensions of the fluid containment structures, alignment and bonding steps in photonic crystal-based sensors present significant practical challenges, and thus add to the overall cost and complexity of fabrication of these devices. First, the components of photonic crystal biosensors are optimally aligned such that the maximum extent of active area of the photonic crystal is exposed to fluid held in the fluid containment structure. Second, bonding and alignment must effectively prevent liquid from exiting a given fluid containment structure and spreading to one or more adjacent fluid containment structures in a multichannel sensor configuration. This requirement is necessary to avoid sensing interferences arising from cross talk between adjacent photonic crystal sensors. Third, the force applied to the photonic crystal structure during alignment and bond must be sufficiently low so as not to damage the nanoscale periodic features of the crystal. Damage to such features can introduce unwanted defect structures to the photonic crystal that can strongly influence sensing capabilities and readout of the device.